RESEARCH ARTICLE PRIMA1 mutation: a new cause of nocturnal frontal lobe epilepsy Michael S. Hildebrand1, Rick Tankard2, Elena V. Gazina3, John A. Damiano1, Kate M. Lawrence1, Hans-Henrik M. Dahl1, Brigid M. Regan1, Aiden Eliot Shearer4, Richard J. H. Smith4, Carla Marini5, Renzo Guerrini5, Angelo Labate6,7, Antonio Gambardella6,7, Paolo Tinuper8, Laura Lichetta8, Sara Baldassari8, Francesca Bisulli8, Tommaso Pippucci8, Ingrid E. Scheffer1,9, Christopher A. Reid3, Steven Petrou3, Melanie Bahlo2 & Samuel F. Berkovic1 1Epilepsy Research Centre, Department of Medicine, Austin Health, University of Melbourne, Melbourne, Victoria, Australia 2Bioinformatics Division, The Walter and Eliza Hall Institute, Melbourne, Victoria, Australia 3The Florey Institute for Neuroscience and Mental Health, The University of Melbourne, Melbourne, Victoria, Australia 4Molecular Otolaryngology & Renal Research Laboratories, Department of Otolaryngology-Head and Neck Surgery, University of Iowa Hospitals and Clinics, Iowa City, Iowa 5Pediatric Neurology and Neurogenetics Unit and Laboratories, A. Meyer Children’s Hospital-University of Florence, Florence, Italy 6Institute of Neurology, University Magna Græcia, Catanzaro, Italy 7Institute of Molecular Bioimaging and Physiology of the National Research Council (IBFM-CNR), Germaneto, CZ, Italy 8Medical Genetics Unit, Polyclinic Sant’Orsola-Malpighi and Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy 9Department of Paediatrics, Royal Children’s Hospital, University of Melbourne, Melbourne, Victoria, Australia Correspondence Abstract Samuel F. Berkovic, Epilepsy Research Centre, Objective Level 2, Melbourne Brain Centre, 245 : Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autoso- Burgundy St. Heidelberg, Victoria 3084, mal dominant; some families have nicotinic acetylcholine receptor subunit Australia. Tel: +61 3 9035 7093; Fax: +61 3 mutations. We report a novel autosomal recessive phenotype in a single family 9496 2291; E-mail: [email protected] and identify the causative gene. Methods: Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to Funding Information Results This study was supported by the National identify the mutation. : Linkage analysis using exome sequence data Health and Medical Research Council from two affected and two unaffected subjects showed homozygous linkage (NHMRC) Program Grant (628952) to S. F. peaks on chromosomes 7, 8, 13, and 14 with maximum LOD scores between B., S. P. and I. E. S., an Australia Fellowship 1.5 and 1.93. Exome variant filtering under these peaks revealed that the (466671) to S. F. B, a Practitioner Fellowship affected siblings were homozygous for a novel splice site mutation (c.93+2T>C) (1006110) to I. E. S and a Career in the PRIMA1 gene on chromosome 14. No additional PRIMA1 mutations Development Fellowship (1063799) to M. S. were found in 300 other NFLE cases. The c.93+2T>C mutation was shown to H. M. B. was supported by an Australian PRIMA1 Research Council (ARC) Future Fellowship lead to skipping of the first coding exon of the mRNA using a mini- (FT100100764) and NHMRC Program Grant gene system. Interpretation: PRIMA1 is a transmembrane protein that anchors (APP1054618), and R. T. by an NHMRC acetylcholinesterase (AChE), an enzyme hydrolyzing acetycholine, to membrane Australian Postgraduate Award. This work rafts of neurons. PRiMA knockout mice have reduction of AChE and accumu- was also supported by the Victorian State lation of acetylcholine at the synapse; our minigene analysis suggests that the Government Operational Infrastructure c.93+2T>C mutation leads to knockout of PRIMA1. Mutations with gain of Support and Australian Government NHMRC function effects in acetylcholine receptor subunits cause autosomal dominant IRIISS funding to M. B., and a Telethon Foundation Project GGP13200 to P. T. NFLE. Thus, enhanced cholinergic responses are the likely cause of the severe and T. P. NFLE and intellectual disability segregating in this family, representing the first recessive case to be reported and the first PRIMA1 mutation implicated in Received: 10 April 2015; Revised: 21 May disease. 2015; Accepted: 29 May 2015. Annals of Clinical and Translational Neurology 2015; 2(8): 821–830 doi: 10.1002/acn3.224 ª 2015 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 821 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. PRIMA1 Mutation Causes Epilepsy M. S. Hildebrand et al. Introduction Nocturnal frontal lobe epilepsy (NFLE) is characterized by frequent, sometimes violent, and often brief seizures at night, that usually commence during childhood. It can arise sporadically or be inherited in an autosomal domi- nant fashion (ADNFLE).1 Clinical features associated with nocturnal seizures include vocalizations, complex and often violent automatisms, and ambulation, making the condition sometimes difficult to distinguish from para- somnias.2 Electroencephalography (EEG) is often unre- vealing, and magnetic resonance imaging (MRI) typically shows no lesions in patients with NFLE. In some cases NFLE is accompanied by intellectual disability and psy- chiatric disorders, the pathogenic mechanisms of which – remain unclear.2 4 Figure 1. Pedigree of Australian family. Two-generation Australian A minority of ADNFLE cases are due to mutations in family of Italian origin segregating ARNFLE (autosomal recessive CHRNA2 CHRNA4 CHRNB2 nocturnal frontal lobe epilepsy) and intellectual disability showing three genes ( , and ) that + a a b genotypes of the PRIMA1 c.93 2 nucleotide. Open symbols encode the 2, 4 and 2 subunits of the neuronal nico- unaffected; shaded symbols affected; double line consanguineous 5–7 tinic acetylcholine receptor. More recently, mutations event; diagonal line deceased. A breast tissue sample was available 8 in the sodium-activated potassium channel KCNT1 were from individual I:2 for genotyping by Sanger sequencing; blood shown to cause a small number of severe familial and samples were obtained from the other family members (I:1, II-1, II-2 sporadic cases. Mutations in the mTOR signaling protein and II-3), and these samples were exome sequenced. DEPDC5 may also present with NFLE, including in 13% – of families in one study.9 12 The etiology of other familial For re-sequencing experiments, 300 sporadic patients NFLEs remain to be elucidated, but additional genes are diagnosed with NFLE were collected and phenotyped expected to be involved. Gain of function of mutated nic- including 212 cases with Italian ancestry. otinic cholinergic receptors appears to be the common mechanism in in vitro studies of human mutations.5,13 Patient samples Based on this electrophysiological data, and the genetic data above, other proteins of the cholinergic nervous sys- Whole blood was obtained and genomic DNA extracted tem may be involved in the pathogenesis of NFLE. using a Qiagen QIAamp DNA Maxi Kit (Valencia, CA). Herein we report a small Australian family of Italian For individual I:2, who died of breast cancer, paraffin- origin segregating autosomal recessive NFLE (ARNFLE) embedded breast tissue was available for genotyping. The and intellectual disability. Using a methodology we paraffin was removed by treatment with xylene and DNA recently reported14 we performed linkage analysis with extracted using phenol chloroform as described previ- whole exome sequencing data to map the disorder to ously.15 Fresh whole blood samples were obtained from multiple genomic loci, and then, using the same sequence affected individual II:3 and her unaffected brother II:1 to data, we identified the causative gene mutation on chro- generate a lymphoblastoid cell line (LCL) for transcript mosome 14. The mutated gene, PRIMA1, encodes a pro- studies. line-rich transmembrane protein that efficiently transforms secreted acetylcholinesterase (AChE) into an Whole exome sequencing enzyme anchored on the outer cell surface. Exome sequencing was performed using 3 lg of venous Materials and Methods blood-derived genomic DNA from each of four family members (I:1, II-1, II-2 and II-3; Fig. 1). Genomic DNA was sonicated to approximately 200 base pair (bp) frag- Family and sporadic cases ments and adaptor-ligated to make a library for paired- A two-generation Australian family of Italian origin with end sequencing. Following amplification and barcoding, NFLE and intellectual disability was studied (Fig. 1). The the libraries were hybridized to biotinylated complemen- Human Research Ethics Committee of Austin Health, tary RNA oligonucleotide baits from the SureSelect Melbourne, Australia, approved this study. Informed con- Human All Exon 50 Mb Kit (Agilent Technologies, sent was obtained from living subjects or their relatives. Santa Clara, CA) and purified using streptavidin-bound 822 ª 2015 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. M. S. Hildebrand et al. PRIMA1 Mutation Causes Epilepsy magnetic beads as described previously.16 Amplification Table 1. Oligonucleotides used in this study. was conducted prior to sequencing on the Illumina HiSeq Target Oligonucleotide (50-30) 2000 system